haematologica 2004; 89(7):July 2004

Letters to the Editor

863

Red Cell Disorders

Childhood sickle cell crises: clinical severity, inflammatorymarkers and the role of interleukin-8

There is emerging consensus that a pro-inflammatorycondition contributes to the vaso-occlusive complica-tions of sickle cell disease (SCD).1-4 We evaluated thepotential value of inflammatory mediators as earlymarkers of severity of painful vaso-occlusive crises(VOC) in SCD. We assayed the plasma levels of cytokines,soluble vascular cell adhesion molecule-1, acute phaseproteins, secretory phospholipase and standard hemato-logic indices.

haematologica 2004; 89:863-864

(http://www.haematologica.org/2004/7/863)

We analyzed 58 VOC that developed in 34 childrenwith sickle cell anemia (Hb SS); 23 of these children werealso studied during steady state. The median age of thepatients was 8.6 years (range: 1 to 18 years). Clinicaldata and samples were systematically collected at admis-sion (day 1), 24 hours later (day 2) and in the steadystate, i.e. at least two months away from any acuteepisode. A control group of 55 healthy normal children(Hb AA), matched for age, sex and race with the SCDgroup, was also studied. Full written consent was

obtained prior to the subjects’ recruitment in the study,which was approved by the local ethical committee.Biological evaluations included (i) hematologic parame-ters (STKS electronic cell counter); (ii) inflammatorymarkers: C-reactive protein (CRP) (nephelometric assay,Berhing BNII analyzer) and α1-glycoprotein (radialimmunodiffusion, Berhing BN100 analyzer), (iii) solublevascular cell adhesion molecule-1 (sVCAM-1), (iv) secre-tory phospholipase A2 (sPLA2), and (v) cytokines: inter-leukin-6 and interleukin-8 (ELISA, according to the man-ufacturers’ instructions). For the purpose of this study anEpisode Severity Index (ESI) was defined according to fiveobjective data (Table 1). In parallel, a clinical level ofseverity was attributed independently by two physiciansto each episode. Severity 1 corresponded to mild episodeswhich could have been treated at home or in a day-carecenter, severity 2 to moderate or severe episodes thatwere never a risk to life and severity 3 to severe and life-threatening episodes. EpiInfo 6.04 software was used forunivariate data analysis. The significance of results wasevaluated by non-parametric Mann-Whitney or Kruskal-Wallis tests. Statistical significance was set at p < 0.05.

Severities 1, 2 and 3 were significantly related to eachparameter included in the ESI (Table 1): zenith of painquotation (p=0.0003), analgesic level (p < 10-6), level 3analgesia duration (p < 10-6), episode duration (p < 10-6)and patient’s behavior during episode (p < 10-6).Moreover, the mean value of the ESI, calculated by

Figure 1. Symptomatic sickle cell patients: variation in biological parameters during steady state and vaso-occlu-sive crises (VOC) according to clinical severity. VOC (A): significant differences on days 1 and 2, VOC (B): Significantdifferences on day 2. Significant differences between: *severity groups 1 and 2, **severity groups 1 and 3,***severity groups 2 and 3. The p values are indicated below each symbol. Significant differences from the steadystate are represented by the columns with a bold border.

Steady state

IL-8pg/mL

0.010.002

0.02

0.0005

0.03

0.03

0.02

0.006

0.0003

0.01

0.01

Leukocytes 109/L

CRPmg/mL

Neutrophils 109/L

VOC (A)

Steady state

Steadystate

17.9(0-255)

31.7(0-480)

38.2(0-352)

61.9(0-492)

68.5(6.3-146)

12.9(7.3-30.3)

14.6(8.9-23.8)

18.3(11.7-25.5)

17.4(12.2-31.2)

13.2(4.3-26.2)

6.7(2.1-38.3)

33.4(3.2-149)

70.3(3.2-255)

73.5(3.4-166)

5.2(1.1-10.3)

6.4(1.8-11.8)

9.7(3.4-15.5)

11.5(3.8-18.9)

15.8(11.3-23.7)

18.2(12.6-25.1)

56.1(0-360)

105(0-525)

−-

−-

−-

−- −- −- −-

−-

−-

−-

−-

−-

−-−-

−-

−-

−-

Steadystate

Steadystate

Steadystate

Day 1 Day 2 Day 2 Day 2Day 2Day 1

Severity 1

Severity 2

Severity 3

VOC (B)

Severity 2Severity 1 Severity 3

120

100

80

60

40

20

0

120

100

80

60

40

20

0

haematologica 2004; 89(7):July 2004864

adding the parameter values (ranging from 4 to 14), wassignificantly different in the severity groups 1, 2 and 3,with respectively mean ESI values of, respectively, 5.0, 8.5and 12.0 (p < 10-6). These data allowed the validation ofthe clinical severity stratification and its use in the fur-ther analysis to reduce the number of comparisons made.

The biological data obtained during the VOC werestudied according to the clinical severity and were com-pared to the steady-state values. Of the 58 VOC analyzed,39.6% were severity 1 episodes, 34.5% severity 2episodes and 25.9% severity 3 episodes; there were nodeaths. Only significant variations related to the studiedcriteria are presented in Figure 1. As can be see from thisfigure, higher IL-8 levels and whole leukocyte countswere present early in severity 3 rather than in severity 1or 2 episodes. Figure 1B, on the other hand, shows thatneutrophils and CRP increased significantly in the severeepisodes but in a delayed manner.

The levels of these biological parameters were statisti-cally higher during severity 2 and 3 episodes than duringthe steady state (Figures 1A and B). IL-6, sVCAM-1 andsPLA2 levels were not significantly different between the3 severity groups.

It is worth noting that the steady state data of the HbSS patients, compared with those of the Hb AA controls,revealed higher leukocyte and neutrophil counts as wellas higher CRP, α1-glycoprotein and sVCAM-1 levels (datanot shown).

These results demonstrate, for the first time, that IL-8is implicated in the severity of VOC episodes. These datacould explain why enhanced serum levels have beenfound in acute chest syndrome5 and enhanced6,7 or unde-tectable serum levels in patients with VOC.5,8 IL-8 maycontribute to SCD pathology through this cytokine’s abil-ity to activate neutrophils, which are the first inflamma-tory cells to appear at the site of vessel damage, exacer-bating and propagating inflammation. Moreover, IL-8might also increase the adherence of sickle red cells toendothelium by activating the α4β1 integrin receptorson sickle reticulocytes.9 The novel therapeutic approach-es designed to inhibit the nuclear factor NF-κB,10 whichpromotes the expression of a number of genes includingthose for IL-8 and IL-6, deserve further development.

Maryse Etienne-Julan,*§ Marie-Sylvaine Belloy,+§

Monique Decastel,§ Samuel Dougaparsad,§ Sylvie Ravion,§

Marie-Dominique Hardy-Dessources§

*Centre Caribéen de la Drépanocytose “Guy Mérault”,§UMR S 458 Inserm-Université des Antilles et de la Guyane,

+Service de Pédiatrie, Centre Hospitalier Universitaire (CHU)Pointe-à-Pitre, Guadeloupe

Funding: This work was supported in part by the CHUPointe-à-Pitre, and by the Institut National de la Santéet de la Recherche Médicale (Inserm UMR S 458).Keywords: sickle cell disease, crisis, inflammation, interleukin,severity marker.Correspondence: Dr. Marie-Dominique Hardy-Dessources, PhD,UMR S 458 Inserm-Université des Antilles et de la Guyane, CHU,97159, Pointe-à-Pitre, Guadeloupe, France. Phone: international+ 590.590.834899. Fax: international + 590.590830513.E-mail: martigua2000@yahoo.fr

References

1. Buchanan GR, Glader BE. Leukocyte counts in children withsickle cell disease. Comparative values in the steady state, vaso-occlusive crisis, and bacterial infection. Am J Dis Child 1978;132:396-8.

2. Makis AC, Hatzimichael EC, Bourantas KL. The role of cytokinesin sickle cell disease. Ann Hematol 2000;79:407-13.

3. Platt OR. Sickle cell anemia as an inflammatory disease. J ClinInvest 2000;106:337-8.

4. Belcher JD, Bryant CJ, Nguyen J, Bowlin PR, Kielbik MC, BischofJC, et al. Transgenic mice have vascular inflammation. Blood2003;101:3953-9.

5. Abboud MR, Taylor EC, Habib D, Dantzler-Johnson T, JacksonSM, Xu F. Elevated serum and bronchroalveolar lavage fluid lev-els of interleukin 8 and granulocyte colony-stimulating factorassociated with the acute chest syndrome in patients with sick-le cell disease. Brit J Haematol 2000;111:482-90.

6. Duits AJ, Schnog JB, Lard LR, Saleh AW, Rojer RA. Elevated IL-8levels during sickle cell crisis. Eur J Haematol 1998;61:302-5.

7. Goncalves MS, Queiroz IL, Cardoso SA, Zanetti A, Strapazoni AC,Adorno E, et al. Interleukin 8 as a vaso-occlusive marker inBrazilian patients with sickle cell disease. Braz J Med Biol Res2001; 34:1309-13.

8. Graido-Gonzales E, Doherty JC, Bergreen EW, Organ G, Telfer M,McMillen MA. Plasma endothelin-1, cytokine and prostaglandinE2 levels in sickle cell disease and acute vaso-occlusive sicklecrisis. Blood 1998;92:2551-5.

9. Kumar A, Eckman JR, Swerlick RA, Wick TM. Phorbol ester stim-ulation increases sickle erythrocyte adherence to endothelium:a novel pathway involving alpha 4 beta 1 integrin receptors onsickle reticulocytes and fibronectin. Blood 1996;88:4348-58.

10. Solovey AA, Solovey AN, Harkness J, Hebbel RP. Modulation ofendothelial cell activation in sickle cell disease: a pilot study.Blood 2001;97:1937-41.

Letters to the Editor

Table 1. Episode Severity Index (ESI): severity stratification according to five clinical parameters.

Zenith of pain Analgesic Level 3 Episode Behaviorquotation type* analgesic duration duration

< 40 = 1 Level 1 = 1 <24h =1 0 to 4 days =1 normal = 041 to 60 = 2 Level 2 = 2 24 to 72h = 2 4 to 7 days = 2 agitated = 161 to 100 = 3 Level 3 = 3 >72h = 3 >7 days = 3 very disturbed or too quiet = 2

*according to the WHO analgesic ladder, i.e.: Level 1: acetaminophen and acetylsalicylic acid or NSAID (ibuprofen); Level 2: 1 + codeine or nalbufine;Level 3: 1 + opioids (e.g. morphine) with or without patient-controlled analgesia.